Miniature inverted-repeat transposable elements (MITEs) are widely distributed in the plant genome and can be methylated. However, whether DNA methylation of MITEs is associated with induced allelic expression and drought tolerance is unclear. Here, we identified the drought-inducible MdRFNR1 (root-type ferredoxin-NADP⁺ oxidoreductase) gene in apple (Malus domestica). MdRFNR1 plays a positive role in drought tolerance by regulating the redox system, including increasing NADP⁺ accumulation and catalase and peroxidase activities and decreasing NADPH levels. Sequence analysis identified a MITE insertion (MITE-MdRF1) in the promoter of MdRFNR1-1 but not the MdRFNR1-2 allele. MdRFNR1-1 but not MdRFNR1-2 expression was significantly induced by drought stress, which was positively associated with the MITE-MdRF1 insertion and its DNA methylation. The methylated MITE-MdRF1 is recognized by the transcriptional anti-silencing factors MdSUVH1 and MdSUVH3, which recruit the DNAJ domain-containing proteins MdDNAJ1, MdDNAJ2, and MdDNAJ5, thereby activating MdRFNR1-1 expression under drought stress. Finally, we showed that MdSUVH1 and MdDNAJ1 are positive regulators of drought tolerance. These findings illustrate the molecular roles of methylated MITE-MdRF1 (which is recognized by the MdSUVH–MdDNAJ complex) in induced MdRFNR1-1 expression as well as the drought response of apple and shed light on the molecular mechanisms of natural variation in perennial trees.

Apple (Malus|$ \times $|domestica) is a popular temperate fruit crop worldwide. However, its growth, productivity, and quality are often adversely affected by abiotic stresses such as drought, extreme temperature, and high salinity. Due to the long juvenile phase and highly heterozygous genome, the conventional breeding approaches for stress-tolerant cultivars are time-consuming and resource-intensive. These issues may be resolved by feasible molecular breeding techniques for apples, such as gene editing and marker-assisted selection. Therefore, it is necessary to acquire a more comprehensive comprehension of the molecular mechanisms underpinning apples’ response to abiotic stress. In this review, we summarize the latest research progress in the molecular response of apples to abiotic stressors, including the gene expression regulation, protein modifications, and epigenetic modifications. We also provide updates on new approaches for improving apple abiotic stress tolerance, while discussing current challenges and future perspectives for apple molecular breeding.

A complex regulatory network governs fruit ripening, but natural variations and functional differentiation of fruit ripening genes remain largely unknown. Utilizing a genome-wide association study (GWAS), we identified the NAC family transcription factor MdNAC18.1, whose expression is closely associated with fruit ripening in apple (Malus × domestica Borkh.). MdNAC18.1 activated the transcription of genes related to fruit softening (Polygalacturonase, PG) and ethylene biosynthesis (1-aminocyclopropane-1-carboxylic acid synthase, ACS), thereby promoting fruit ripening of apple and tomato (Solanum lycopersicum). There were two single-nucleotide polymorphisms (SNP-1,545 and SNP-2,002) and a 58-bp insertion–deletion (InDel-58) in the promoter region of MdNAC18.1. Among these, InDel-58 serves as the main effector in activating the expression of MdNAC18.1 and driving fruit ripening. InDel-58 determines the binding affinity of the class D MADS-box protein AGAMOUS-LIKE 11 (MdAGL11), a negative regulator of fruit ripening. The InDel-58 deletion in the early-ripening genotype reduces the inhibitory effect of MdAGL11 on MdNAC18.1. Moreover, MdNAC18.1 and its homologous genes originated from a common ancestor across 61 angiosperms, with functional diversification attributed to tandem replications that occurred in basal angiosperms. In summary, our study revealed how a set of natural variations influence fruit ripening and explored the functional diversification of MdNAC18.1 during evolution.

Apples (Malus × domestica Borkh.) can be categorized into early-, medium-, and late-ripening cultivars based on the length of the fruit developmental phases. The lengthening of the apple ripening period has a direct impact on its economic worth and market competitiveness, although the underlying mechanism is mostly unclear. In the current study, the development and maturation of the early-ripening ‘Geneva Early’ (GE) and late-ripening ‘Hanfu’ (HF) cultivars of apple fruit were studied using transcriptomics to detect and identify the changes of differential genes. Results showed that the two varieties had different ripening periods, but in both, the development process of fruit ripening required cell division, cell expansion, starch accumulation, and secondary metabolite accumulation. In the early stages of fruit development (G1 to G2), the GE’s fruit size was larger than HF’s, and the GO analysis revealed an enrichment in genes involved in the metabolism of fatty acids and carbon molecules. In G2 phase, the GE involved numerous regulatory factors of hormonal pathways, while in HF this phase was mainly enriched in the metabolism of sugars and carbohydrates. The results indicated that during GE development, the relevant genes regulating fruit development were expressed earlier than HF, which made fruit development enter the next development phase earlier, thereby shortening the fruit development phase. These findings contributed to an improved understanding of the molecular basis of apple ripening and provide a reliable reference for apple breeding using genomics.

Fruit ripening is governed by a complex regulatory network, and ethylene plays an important role in this process. MdKING1 is a γ subunit of SNF1-related protein kinases (SnRKs), but the function was unclear. Here, we characterized the role of MdKING1 during fruit ripening, which can promote fruit ripening through the ethylene pathway. Our findings reveal that MdKING1 has higher expression in early-ripening cultivars than late-ripening during the early stage of apple fruit development, and its transcription level significantly increased during apple fruit ripening. Overexpression of MdKING1 (MdKING1 OE) in tomatoes could promote early ripening of fruits, with the increase in ethylene content and the loss of fruit firmness. Ethylene inhibitor treatment could delay the fruit ripening of both MdKING1 OE and WT fruits. However, MdKING1 OE fruits turned fruit ripe faster, with an increase in carotenoid content compared with WT. In addition, the expression of genes involved in ethylene biosynthesis (SlACO1, SlACS2, and SlACS4), carotenoid biosynthesis (SlPSY1 and SlGgpps2a), and fruit firmness regulation (SlPG2a, SlPL, and SlCEL2) was also increased in the fruits of MdKING1 OE plants. In conclusion, our results suggest that MdKING1 plays a key role in promoting tomato fruit ripening, thus providing a theoretical basis for apple fruit quality improvement by genetic engineering in the future.